The opioid receptors are a subfamily of the family A G protein-coupled opioid receptor superfamily and consist of mu (OPRM1), delta (OPRD1), and kappa (OPRK1), all of which activate inhibitory G proteins. The dynorphins act as endogenous agonists of OPRK to activate a variety of signaling transduction pathways including those involving mitogen activated protein kinases (MAPK). Activation of OPRK leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward. Hence, OPRK antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists are very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK antagonists have been developed. New OPRK antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK- dynorphin system and as potential pharmacotherapies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports ML350 as a highly potent OPRK1 antagonist with an IC50 of 9-16 nM, with high selectivity (selectivities vs. OPRD1 and OPRM1 of 219-382–fold and 20-35–fold, respectively). ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK agonist in a tail flick assay in mice. ML350 serves as a novel OPRK antagonist that can be developed as a therapeutic for the treatment of a variety of disorders involving the OPRK1-dynorphin system.